Piezoelectric Stimulation Induces Osteogenesis in Mesenchymal Stem Cells Cultured on Electroactive Two-Dimensional Substrates

Physical cues have been shown to be effective in inducing osteogenic differentiation of mesenchymal stem cells (MSCs). Here, we propose piezoelectric stimulation as a potential osteogenic cue mimicking the electroactive properties of bone's extracellular matrix. When combined with a magnetostrictive component, piezoelectric polymers can be used for MSC stimulation by applying an external magnetic field. The deformation of the magnetostrictive component produces a deformation in the polymer matrix, generating a change in the surface charge that induces an electric field that can be transmitted to the cells. Cell adhesion, cytoskeleton changes, and metabolomics are the first evidence of MSC osteoblastogenesis and can be used to study initial MSC response to this kind of stimulation. In the current study, poly(vinylidene) fluoride (PVDF) piezoelectric films with and without cobalt ferrite oxide (CFO) crystallized from the melt in the presence of the ionic liquid 1-butyl-3-methyl-imidazolium chloride ([Bmim][Cl]) were produced. [Bmim][Cl] allowed the production of the β-phase, the most electroactive phase, even without CFO. After ionic liquid removal, PVDF and PVDF-CFO films presented high percentages of the β-phase and similar crystalline content. Incorporating CFO nanoparticles was effective, allowing the electromechanical stimulation of MSCs by applying a magnetic field with a bioreactor. Before stimulation, the initial response of MSCs was characterized in static conditions, showing that the produced films were biocompatible and noncytotoxic, allowing MSC adhesion and proliferation in the short term. Stimulation experiments revealed that MSCs electromechanically stimulated for 3 days in PVDF-CFO supports showed longer focal adhesions and decreased vimentin cytoskeletal density, both signals of early osteogenic differentiation. Furthermore, they rearranged their energy metabolism toward an osteogenic phenotype after 7 days of culture under the same stimulation. The results prove that MSCs respond to electromechanical stimulation by osteogenic differentiation.